Vitronectin receptor antagonist

ABSTRACT

A compound of the formula (I) is disclosed which is a vitronectin receptor antagonist and is useful in the treatment of osteoporosis:                    
     or a pharmaceutically acceptable salt thereof.

This application is a 371 of PCT/US99/28662 filed Dec. 03, 1999 whichclaims the benefit of No. 60/110,903 filed Dec. 04, 1998.

FIELD OF THE INVENTION

This invention relates to a pharmaceutically active compound whichinhibits the vitronectin receptor and is useful for the treatment ofinflammation, cancer and cardiovascular disorders, such asatherosclerosis and restenosis, and diseases wherein bone resorption isa factor, such as osteoporosis.

BACKGROUND OF THE INVENTION

Integrins are a superfamily of cell adhesion receptors, which aretransmembrane glycoproteins expressed on a variety of cells. These cellsurface adhesion receptors include gpIIb/IIIa (the fibrinogen receptor)and α_(v)β₃ (the vitronectin receptor). The fibrinogen receptorgpIIb/IIIa is expressed on the platelet surface, and mediates plateletaggregation and the formation of a hemostatic clot at the site of ableeding wound. Philips, et al., Blood., 1988, 71, 831. The vitronectinreceptor α_(v)β₃ is expressed on a number of cells, includingendothelial, smooth muscle, osteoclast, and tumor cells, and, thus, ithas a variety of functions. The α_(v)β₃ receptor expressed on themembrane of osteoclast cells mediates the adhesion of osteoclasts to thebone matrix, a key step in the bone resorption process. Ross, et al., J.Biol. Chem., 1987, 262, 7703. A disease characterized by excessive boneresorption is osteoporosis. The α_(v)β₃ receptor expressed on humanaortic smooth muscle cells mediates their migration into neointima, aprocess which can lead to restenosis after percutaneous coronaryangioplasty. Brown, et al., Cardiovascular Res., 1994, 28, 1815.Additionally, Brooks, et al., Cell, 1994, 79, 1157 has shown that anα_(v)β₃ antagonist is able to promote tumor regression by inducingapoptosis of angiogenic blood vessels. Thus, agents that block thevitronectin receptor would be useful in treating diseases, such asosteoporosis, restenosis and cancer.

The vitronectin receptor is now known to refer to three differentintegrins, designated α_(v)β₁, α_(v)β₃ and α_(v)β₅. Horton, et al., Int.J. Exp. Pathol., 1990, 71, 741. α_(v)β₃ binds fibronectin andvitronectin. α_(v)β₃ binds a large variety of ligands, including fibrin,fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand'sfactor, osteopontin and bone sialoprotein I. α_(v)β₅ binds vitronectin.The vitronectin receptor α_(v)β₅ has been shown to be involved in celladhesion of a variety of cell types, including microvascular endothelialcells, (Davis, et al., J. Cell. Biol., 1993, 51, 206), and its role inangiogenesis has been confirmed. Brooks, et al., Science, 1994, 264,569. This integrin is expressed on blood vessels in human woundgranulation tissue, but not in normal skin.

The vitronectin receptor is known to bind to bone matrix proteins whichcontain the tri-peptide Arg-Gly-Asp (or RGD) motif. Thus, Horton, etal., Exp. Cell Res. 1991, 195, 368, disclose that RGD-containingpeptides and an anti-vitronectin receptor antibody (23C6) inhibitdentine resorption and cell spreading by osteoclasts. In addition, Sato,et al., J. Cell Biol. 1990, 111, 1713 discloses that echistatin, a snakevenom peptide which contains the RGD sequence, is a potent inhibitor ofbone resorption in tissue culture, and inhibits attachment ofosteoclasts to bone.

It has now been discovered that a certain compound is a potent inhibitorof the α_(v)β₃ and α_(v)β₅ receptors. In particular, it has beendiscovered that such compound is more potent inhibitors of thevitronectin receptor than the fibrinogen receptor.

SUMMARY OF THE INVENTION

This invention comprises a compound of the formula (I) as describedhereinafter, which has pharmacological activity for the inhibition ofthe vitronection receptor and is useful in the treatment ofinflammation, cancer and cardiovascular disorders, such asatherosclerosis and restenosis, and diseases wherein bone resorption isa factor, such as osteoporosis.

This invention is also a pharmaceutical composition comprising acompound according to formula (I) and a pharmaceutically carrier.

This invention is also a method of treating diseases which are mediatedby the vitronectin receptor. In a particular aspect, the compound ofthis invention is useful for treating atherosclerosis, restenosis,inflammation, cancer and diseases wherein bone resorption is a factor,such as osteoporosis.

DETAILED DESCRIPTION

This invention comprises a novel compound which is a more potentinhibitor of the vitronectin receptor than the fibrinogen receptor. Thenovel compound comprises a dibenzocycloheptene core in which anitrogen-containing substituent is present on one of the aromaticsix-membered rings of the dibenzocycloheptene and an aliphaticsubstituent containing an acidic moiety is present on the seven-memberedring of the dibenzocycloheptene. The dibenzocycloheptene ring system isbelieved to orient the substituent sidechains on the six and sevenmembered rings so that they may interact favorably with the vitronectinreceptor. It is preferred that about twelve to fourteen interveningcovalent bonds via the shortest intramolecular path will exist betweenthe acidic group on the aliphatic substituent of the seven-membered ringof the dibenzocycloheptene and the nitrogen of the nitrogen-containingsubstituent on one of the aromatic six-membered ring of thedibenzocycloheptene.

This invention comprises a compound of formula (I):

or a pharmaceutically acceptable salt thereof.

The compound of formula (I) inhibits the binding of vitronectin andother RGD-containing peptides to the vitronectin receptor. Inhibition ofthe vitronectin receptor on osteoclasts inhibits osteoclastic boneresorption and is useful in the treatment of diseases wherein boneresorption is associated with pathology, such as osteoporosis andosteoarthritis.

In another aspect, this invention is a method for stimulating boneformation which comprises administering a compound of formula (I) whichcauses an increase in osteocalcin release. Increased bone production isa clear benefit in disease states wherein there is a deficiency ofmineralized bone mass or remodeling of bone is desired, such as fracturehealing and the prevention of bone fractures. Diseases and metabolicdisorders which result in loss of bone structure would also benefit fromsuch treatment. For instance, hyperparathyroidism, Paget's disease,hypercalcemia of malignancy, osteolytic lesions produced by bonemetastasis, bone loss due to immobilization or sex hormone deficiency,Behcet's disease, osteomalacia, hyperostosis and osteopetrosis, couldbenefit from administering a compound of this invention.

Additionally, since the compound of the instant invention inhibitsvitronectin receptors on a number of different types of cells, saidcompound would be useful in the treatment of inflammatory disorders,such as rheumatoid arthritis and psoriasis, and cardiovascular diseases,such as atherosclerosis and restenosis. The compound of Formula (I) ofthe present invention may be useful for the treatment or prevention ofother diseases including, but not limited to, thromboembolic disorders,asthma, allergies, adult respiratory distress syndrome, graft versushost disease, organ transplant rejection, septic shock, eczema, contactdermatitis, inflammatory bowel disease, and other autoimmune diseases.The compound of the present invention may also be useful for woundhealing.

The compound of the present invention is also useful for the treatment,including prevention, of angiogenic disorders. The term angiogenicdisorders as used herein includes conditions involving abnormalneovascularization. Where the growth of new blood vessels is the causeof, or contributes to, the pathology associated with a disease,inhibition of angiogenisis will reduce the deleterious effects of thedisease. An example of such a disease target is diabetic retinopathy.Where the growth of new blood vessels is required to support growth of adeleterious tissue, inhibition of angiogenisis will reduce the bloodsupply to the tissue and thereby contribute to reduction in tissue massbased on blood supply requirements. Examples include growth of tumorswhere neovascularization is a continual requirement in order that thetumor grow and the establishment of solid tumor metastases. Thus, thecompound of the present invention inhibit tumor tissue angiogenesis,thereby preventing tumor metastasis and tumor growth.

Thus, according to the methods of the present invention, the inhibitionof angiogenesis using the compound of the present invention canameliorate the symptoms of the disease, and, in some cases, can cure thedisease.

Another therapeutic target for the compound of the instant invention iseye diseases chacterized by neovascularization. Such eye diseasesinclude corneal neovascular disorders, such as corneal transplantation,herpetic keratitis, luetic keratitis, pterygium and neovascular pannusassociated with contact lens use. Additional eye diseases also includeage-related macular degeneration, presumed ocular histoplasmosis,retinopathy of prematurity and neovascular glaucoma.

This invention further provides a method of inhibiting tumor growthwhich comprises administering stepwise or in physical combination acompound of formula (I) and an antineoplastic agent, such as topotecanand cisplatin.

The novel compound of this invention is(S)-10,11-dihydro-3-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethoxy]-5H-dibenzo[a,d]cycloheptene-10-aceticacid or a pharmaceutically acceptable salt thereof.

According to the present invention, the (S) configuration of the formula(I) compound is preferred.

Also included in this invention are prodrugs of the compound of thisinvention. Prodrugs are considered to be any covalently bonded carrierswhich release the active parent drug according to formula (I) in vivo.Thus, in another aspect of this invention are novel prodrugs, which arealso intermediates in the preparation of the formula (I) compound, offormula (II):

or a pharmaceutically acceptable salt thereof.

Abbreviations and symbols commonly used in the peptide and chemical artsare used herein to describe the compound of this invention. In general,the amino acid abbreviations follow the IUPAC-IUB Joint Commission onBiochemical Nomenclature as described in Eur. J. Biochem., 158, 9(1984).

C₁₋₆alkyl as applied herein means an optionally substituted alkyl groupof 1 to 6 carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, t-butyl, pentyl, n-pentyl, isopentyl, neopentyl andhexyl and the simple aliphatic isomers thereof.

Any C₁₋₆ alkyl may be optionally substituted with the group R^(x), whichmay be on any carbon atom that results in a stable structure and isavailable by conventional synthetic techniques. Suitable groups forR^(x) are C₁₋₄alkyl, OR″, SR″, C₁₋₄alkylsulfonyl, C₁₋₄alkylsulfoxyl,—CN, N(R″)₂, CH₂N(R″)₂, —NO₂, —CF₃, —CO₂R″, —CON(R″)₂, —COR″,—NR″C(O)R″, F, Cl, Br, I, or CF₃S(O)_(r)—, wherein r is 0, 1 or 2 and R″is H or C₁₋₆alkyl.

Certain radical groups are abbreviated herein. t-Bu refers to thetertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical,Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to thephenyl radical, Cbz refers to the benzyloxycarbonyl radical, Bn refersto the benzyl radical, Me refers to methyl, Et refers to ethyl, Acrefers to acetyl, Alk refers to C₁₋₄alkyl, Nph refers to 1- or2-naphthyl and cHex refers to cyclohexyl. Tet refers to 5-tetrazolyl.

Certain reagents are abbreviated herein. DCC refers todicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, DIEArefers to diisopropylethyl amine, EDC refers to1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride. HOBtrefers to 1-hydroxybenzotriazole, THP refers to tetrahydrofuiran, DIEArefers to diisopropylethylamine, DEAD refers to diethylazodicarboxylate, PPh₃ refers to triphenylphosphine, DIAD refers todiisopropyl azodicarboxylate, DME refers to dimethoxyethane, DMF refersto dimethylformamide, NBS refers to N-bromosuccinimide, Pd/C refers to apalladium on carbon catalyst, PPA refers to polyphosphoric acid, DPPArefers to diphenylphosphoryl azide, BOP refers tobenzotriazol-1-yloxy-tris(dimethyl-amino)phosphoniumhexafluorophosphate, HF refers to hydrofluoric acid, TEA refers totriethylamine, TFA refers to trifluoroacetic acid, PCC refers topyridinium chlorochromate.

Compound of the formula (I) may be prepared by the methods described inBondinell et al., PCT Publication No. WO 97/01540 (InternationalApplication No. PCT/US96/11108), published Jan. 16, 1997, the entiredisclosure of which is incorporated herein by reference.

Additionally, the compound of formula (I) is prepared by methodsanalogous to those described in the schemes that are detailedhereinafter.

a) 10% Pd/C, HOAc; b) SOCl₂, toluene; c) AlCl₃, CH₂Cl₂

Scheme I details the preparation of an intermediate useful in thepreparation of formula (I) compound.

a) LiN(TMS)₂, ethyl bromoacetate; b) Jones reagent, OsO₄; c) H₂, 10%Pd/C, HOAC; d) C₂O₂Cl₂, DMF; e) AlCl₃, CH₂Cl₂, RT; f) H₂, 10% Pd/C, HOAC

Scheme II also details the preparation of an intermediate useful in thepreparation of formula (I) compound.

(a) EtOAc/LiHMDS, THF; (b) H₂, 10% Pd/C, conc. HCl, AcOH; (c) EtSH,AlCl₃, CH₂Cl₂; (d)2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethanol, diisopropylazodicarboxylate, (Ph)₃P; (e) 1.0 N LiOH, EtOH; HCl.

Scheme III details the preparation of a formula (I) compound. Reactionof III-1 (which is a Scheme I-3 compound) in an aldol-type reaction withthe enolate of ethyl acetate, which can be generated from ethyl acetateon exposure to an appropriate amide base, for instance lithiumdiisopropylamide (LDA) or lithium bis(trimethylsilyl)amide (LiHMDS),gives III-2. Frequently. THF is the solvent of choice for an aldolreaction, although THF in the presence of various additives, forinstance HMPA or TMEDA, is often used. Reduction of III-2 to give III-3(which is a Scheme II-6 compound) can be accomplished by hydrogenolysisover an appropriate catalyst, for example palladium metal on activatedcarbon. (Pd/C), in an appropriate solvent, such as acetic acid, in thepresence of a mineral acid such as HCl. Alternatively, this reductioncan be accomplished by treatment of III-2 with triethylsilane in thepresence of boron trifluoride etherate by the general method ofOrfanopoulos and Smonou (Synth. Commun. 1988, 833). Removal of themethyl ether of III-3 to give III-4 can be accomplished with BBr₃ in aninert solvent, for example CH₂Cl₂, or by reaction with ethanethiol andAlCl₃ in an inert solvent, preferably CH₂Cl₂. Other useful methods forremoval of a methyl ether are described in Greene, “Protective Groups inOrganic Synthesis” (published by John Wiley and Sons). Compound 4 ofScheme 3 (III-4) is reacted with2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethanol to afford III-5.The reaction is mediated by the complex formed between diisopropylazodicarboxylate and triphenylphosphine, and is conducted in an aproticsolvent, for instance THF, CH₂Cl₂, or DMF. The ethyl ester of III-5 ishydrolyzed using aqueous base, for example, LiOH in aqueous THF or NaOHin aqueous methanol or ethanol, and the intermediate carboxylate salt isacidified with a suitable acid, for instance TFA or HCl, to afford thecarboxylic acid III-6. Alternatively, the intermediate carboxylate saltcan be isolated, if desired, or a carboxylate salt of the freecarboxylic acid can be prepared by methods well-known to those of skillin the art.

(a) PhOH, Cu, K₂CO₃; (b) sulfur, morpholine; (c) KOH, H₂O, i-PrOH; (d)SOCl₂, benzene; (e) AlCl₃, CH₂Cl₂; (f) EtOAc, LiN(TMS)₂, TMEDA, THF; (g)Et₃SiH, BF₃. OEt₂, CH₂Cl₂; (h) H₂, Pd/C, EtOH; (i) BBr₃, CH₂Cl₂.

Commercially available 2-fluoro-4-methoxyacetophenone (IV-1) reacts withan alcohol, for example phenol, in the presence of copper metal and asuitable base, for instance K₂CO₃, to afford the diaryl ether IV-2. Ontreatment with sulfur and an appropriate primary or secondary amine,preferably morpholine, according to the general method of Harris (J.Med. Chem. 1982, 25, 855), IV-2 is converted to IV-3 in a classicalWillgerodt-Kindler reaction. The thioamide thus obtained is hydrolyzedto the corresponding carboxylic acid IV-4 by reaction with an alkalimetal hydroxide, suitably KOH, in an aqueous alcoholic solvent, such asaqueous MeOH, EtOH, or i-PrOH. Carboxylic acid IV-4 is converted to thecorresponding acid chloride by reaction with either SOCl₂ or oxalylchloride according to conditions well-known to those of skill in theart. Treatment of this acid chloride with an appropriate Friedel-Craftscatalyst, such as AlCl₃ or SnCl₄, in an inert solvent, such as CH₂Cl₂ orCS₂, provides the cyclic ketone IV-5. Alternatively, acid IV-4 can beconverted directly to ketone IV-5 under acidic conditions, for examplewith polyphosphoric acid. Reaction of IV-5 in an aldol—type reactionwith the enolate of ethyl acetate, which can be generated from ethylacetate on exposure to an appropriate amide base, for instance lithiumdiisopropylamide (LDA) or lithium bis(trimethylsilyl)amide (LiHMDS),gives IV-6. Frequently, THF is the solvent of choice for an aldolreaction, although THF in the presence of various additives, forinstance HMPA or TMEDA, is often used. Reduction of IV-6 to give IV-7can be accomplished by treatment of IV-6 with triethylsilane in thepresence of boron trifluoride etherate by the general method ofOrphanopoulos and Smonu (Synth. Commun. 1988, 833). Any olefinicby-products that result from elimination of the alcohol are reduced byhydrogenation over an appropriate catalyst, for example palladium metalon activated carbon (Pd/C), in an appropriate solvent, such as MeOH orEtOH. Alternatively, the reduction of IV-6 to give IV-7 can beaccomplished by hydrogenolysis in the presence of a mineral acid such asHCl. Typically, this reaction is catalyzed by Pd/C, and is optimallyconducted in acetic acid. Removal of the methyl ether of IV-7 to giveIV-8 can be accomplished with BBr₃ in an inert solvent, for exampleCH₂Cl₂, or by reaction with ethanethiol and AlCl₃ in an inert solvent,preferably CH₂Cl₂. Other useful methods for removal of a methyl etherare described in Greene, “Protective Groups in Organic Synthesis”(published by John Wiley and Sons). IV-8 is subsequently converted toformula (I) compound following the procedure outlined in Scheme III.

Acid addition salts of the compound are prepared in a standard manner ina suitable solvent from the parent compound and an excess of an acid,such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric,acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain ofthe compound form inner salts or zwitterions which may be acceptable.Cationic salts are prepared by treating the parent compound with anexcess of an alkaline reagent, such as a hydroxide, carbonate oralkoxide, containing the appropriate cation; or with an appropriateorganic amine. Cations such as Li⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺ and NH₄ ⁺ arespecific examples of cations present in pharmaceutically acceptablesalts.

This invention also provides a pharmaceutical composition whichcomprises a compound according to formula (I) and a pharmaceuticallyacceptable carrier. Accordingly, the compound of formula (I) may be usedin the manufacture of a medicament. Pharmaceutical compositions of thecompound of formula (I) prepared as hereinbefore described may beformulated as solutions or lyophilized powders for parenteraladministration. Powders may be reconstituted by addition of a suitablediluent or other pharmaceutically acceptable carrier prior to use. Theliquid formulation may be a buffered, isotonic, aqueous solution.Examples of suitable diluents are normal isotonic saline solution,standard 5% dextrose in water or buffered sodium or ammonium acetatesolution. Such formulation is especially suitable for parenteraladministration, but may also be used for oral administration orcontained in a metered dose inhaler or nebulizer for insufflation. Itmay be desirable to add excipients such as polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol,sodium chloride or sodium citrate.

Alternately, these compound may be encapsulated, tableted or prepared ina emulsion or syrup for oral administration. Pharmaceutically acceptablesolid or liquid carriers may be added to enhance or stabilize thecomposition, or to facilitate preparation of the composition. Solidcarriers include starch, lactose, calcium sulfate dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly p.o.or filled into a soft gelatin capsule.

For rectal administration, the compound of this invention may also becombined with excipients such as cocoa butter, glycerin, gelatin orpolyethylene glycols and molded into a suppository.

The compound described herein are antagonists of the vitronectinreceptor, and are useful for treating diseases wherein the underlyingpathology is attributable to ligand or cell which interacts with thevitronectin receptor. For instance, these compound are useful for thetreatment of diseases wherein loss of the bone matrix creates pathology.Thus, the instant compound are useful for the treatment of ostoeporosis,hyperparathyroidism, Paget's disease, hypercalcemia of malignancy,osteolytic lesions produced by bone metastasis, bone loss due toimmobilization or sex hormone deficiency. The compound of this inventionare also believed to have utility as antitumor, anti-angiogenic,antiinflammatory and anti-metastatic agents, and be useful in thetreatment of atherosclerosis and restenosis.

The compound is administered either orally or parenterally to thepatient, in a manner such that the concentration of drug is sufficientto inhibit bone resorption, or other such indication. The pharmaceuticalcomposition containing the compound is administered at an oral dose ofbetween about 0.1 to about 50 mg/kg in a manner consistent with thecondition of the patient. Preferably the oral dose would be about 0.5 toabout 20 mg/kg. For acute therapy, parenteral administration ispreferred. An intravenous infusion of the peptide in 5% dextrose inwater or normal saline, or a similar formulation with suitableexcipients, is most effective, although an intramuscular bolus injectionis also useful. Typically, the parenteral dose will be about 0.01 toabout 100 mg/kg; preferably between 0.1 and 20 mg/kg. The compound areadministered one to four times daily at a level to achieve a total dailydose of about 0.4 to about 400 mg/kg/day. The precise level and methodby which the compound are administered is readily determined by oneroutinely skilled in the art by comparing the blood level of the agentto the concentration required to have a therapeutic effect.

This invention further provides a method for treating osteoporosis orinhibiting bone loss which comprises administering stepwise or inphysical combination a compound of formula (I) and other inhibitors ofbone resorption, such as bisphosphonates (i.e., allendronate), hormonereplacement therapy, anti-estrogens, or calcitonin. In addition, thisinvention provides a method of treatment using a compound of thisinvention and an anabolic agent, such as the bone morphogenic protein,iproflavone, useful in the prevention of bone loss and/or to increasebone mass.

Additionally, this invention provides a method of inhibiting tumorgrowth which comprises administering stepwise or in physical combinationa compound of formula (I) and an antineoplastic agent. Compound of thecamptothecin analog class, such as topotecan, irinotecan and9-aminocamptothecin, and platinum coordination complexes, such ascisplatin, ormaplatin and tetraplatin, are well known groups ofantineoplastic agents. Compound of the camptothecin analog class aredescribed in U.S. Pat. Nos. 5,004,758, 4,604,463, 4,473,692, 4,545,880,4,342,776, 4,513,138, 4,399,276, EP Patent Application Publication Nos.0 418 099 and 0 088 642, Wani, et al., J. Med. Chem., 1986, 29, 2358,Wani, et al., J. Med. Chem., 1980, 23, 554, Wani, et al., J. Med. Chem,1987, 30, 1774, and Nitta, et al., Proc. 14th International Congr.Chemotherapy., 1985, Anticancer Section 1, 28, the entire disclosure ofeach which is hereby incorporated by reference. The platinumcoordination complex, cisplatin, is available under the name Platinol®from Bristol Myers-Squibb Corporation. Useful formulations for cisplatinare described in U.S. Pat. Nos. 5,562,925 and 4,310,515, the entiredisclosure of each which is hereby incorporated by reference.

In the method of inhibiting tumor growth which comprises administeringstepwise or in physical combination a compound of formula (I) and anantineoplastic agent, the platinum coordination compound, for examplecisplatin, can be administered using slow intravenous infusion. Thepreferred carrier is a dextrose/saline solution containing mannitol. Thedose schedule of the platinum coordination compound may be on the basisof from about 1 to about 500 mg per square meter (mg/m²) of body surfacearea per course of treatment. Infusions of the platinum coordiationcompound may be given one to two times weekly, and the weekly treatmentsmay be repeated several times. Using a compound of the camptothecinanalog class in a parenteral administration, the course of therapygenerally employed is from about 0.1 to about 300.0 mg/m² of bodysurface area per day for about five consecutive days. Most preferably,the course of therapy employed for topotecan is from about 1.0 to about2.0 mg/m² of body surface area per day for about five consecutive days.Preferably, the course of therapy is repeated at least once at about aseven day to about a twenty-eight day interval.

The pharmaceutical composition may be formulated with both the compoundof formula (I) and the antineoplastic agent in the same container, butformualtion in different containers is preferred. When both agents areprovided in solution form, they can be contained in aninfusion/injection system for simultaneous administration or in a tandemarrangement.

For convenient administration of the compound of formula (I) and theantineoplastic agent at the same or different times, a kit is prepared,comprising, in a single container, such as a box, carton or othercontainer, individual bottles, bags, vials or other containers eachhaving an effective amount of the compound of formula (I) for parenteraladministration, as described above, and an effective amount of theantineoplastic agent for parenteral administration, as described above.Such kit can comprise, for example, both pharmaceutical agents inseparate containers or the same container, optionally as lyophilizedplugs, and containers of solutions for reconstitution. A variation ofthis is to include the solution for reconstitution and the lyophilizedplug in two chambers of a single container, which can be caused to admixprior to use. With such an arrangement, the antineoplastic agent and thecompound of this invention may be packaged separately, as in twocontainers, or lyophilized together as a powder and provided in a singlecontainer.

When both agents are provided in solution form, they can be contained inan infusion/injection system for simultaneous administration or in atandem arrangement. For example, the compound of formula (I) may be inan i.v. injectable form, or infusion bag linked in series, via tubing,to the antineoplastic agent in a second infusion bag. Using such asystem, a patient can receive an initial bolus-type injection orinfusion of the compound of formula (I) followed by an infusion of theantineoplastic agent.

The compound may be tested in one of several biological assays todetermine the concentration of compound which is required to have agiven pharmacological effect.

Inhibition of Vitronectin Binding

Solid-Phase [³H]-SK&F-107260 Binding to α_(v)β₃: Human placenta or humanplatelet α_(v)β₃ (0.1-0.3 mg/mL) in buffer T (containing 2 mM CaCl₂ and1% octylglucoside) was diluted with buffer T containing 1 mM CaCl₂, 1 mMMnCl₂, 1 mM MgCl2 (buffer A) and 0.05% NaN₃, and then immediately addedto 96-well ELISA plates (Corning, New York, N.Y.) at 0.1 mL per well.0.1-0.2 μg of α_(v)β₃ was added per well. The plates were incubatedovernight at 4° C. At the time of the experiment, the wells were washedonce with buffer A and were incubated with 0.1 mL of 3.5% bovine serumalbumin in the same buffer for 1 hr at room temperature. Followingincubation the wells were aspirated completely and washed twice with 0.2mL buffer A.

Compound were dissolved in 100% DMSO to give a 2 mM stock solution,which was diluted with binding buffer (15 mM Tris-HCl (pH 7.4), 100 mMNaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂) to a final compoundconcentration of 100 μM. This solution is then diluted to the requiredfinal compound concentration. Various concentrations of unlabeledantagonists (0.001-100 μM) were added to the wells in triplicates,followed by the addition of 5.0 nM of [³H]-SK&F-107260 (65-86 Ci/mmol).

The plates were incubated for 1 hr at room temperature. Followingincubation the wells were aspirated completely and washed once with 0.2mL of ice cold buffer A in a well-to-well fashion. The receptors weresolubilized with 0.1 mL of 1% SDS and the bound [³H]-SK&F-107260 wasdetermined by liquid scintillation counting with the addition of 3 mLReady Safe in a Beckman LS Liquid Scintillation Counter, with 40%efficiency. Nonspecific binding of [³H]-SK&F-107260 was determined inthe presence of 2 μM SK&F-107260 and was consistently less than 1% oftotal radioligand input. The IC₅₀ (concentration of the antagonist toinhibit 50% binding of [³H]-SK&F-107260) was determined by a nonlinear,least squares curve-fitting routine, which was modified from theLUNDON-2 program. The K_(i) (dissociation constant of the antagonist)was calculated according to the equation: K_(i)=IC₅₀/(1+L/K_(d)), whereL and K_(d) were the concentration and the dissociation constant of[³H]-SK&F-107260, respectively.

The compound of the present invention inhibits vitronectin binding toSK&F 107260 at a K_(i) of about 1.7 nanomolar.

Compound of this invention are also tested for in vitro and in vivo boneresorption in assays standard in the art for evaluating inhibition ofbone formation, such as the pit formation assay disclosed in EP 528 587,which may also be performed using human osteoclasts in place of ratosteoclasts, and the ovarectomized rat model, described by Wronski etal., Cells and Materials 1991, Sup. 1, 69-74.

Vascular Smooth Muscle Cell Migration Assay

Rat or human aortic smooth muscle cells were used. The cell migrationwas monitored in a Transwell cell culture chamber by using apolycarbonate membrane with pores of 8 um (Costar). The lower surface ofthe filter was coated with vitronectin. Cells were suspended in DMEMsupplemented with 0.2% bovine serum albumin at a concentration of2.5-5.0×10⁶ cells/mL, and were pretreated with test compound at variousconcentrations for 20 min at 20° C. The solvent alone was used ascontrol. 0.2 mL of the cell suspension was placed in the uppercompartment of the chamber. The lower compartment contained 0.6 mL ofDMEM supplemented with 0.2% bovine serum albumin. Incubation was carriedout at 37° C. in an atmosphere of 95% air/5% CO₂ for 24 hr. Afterincubation, the non-migrated cells on the upper surface of the filterwere removed by gentle scraping. The filter was then fixed in methanoland stained with 10% Giemsa stain. Migration was measured either by a)counting the number of cells that had migrated to the lower surface ofthe filter or by b) extracting the stained cells with 10% acetic acidfollowed by determining the absorbance at 600 nM.

Thyroparathyroidectomized Rat Model

Each experimental group consists of 5-6 adult male Sprague-Dawley rats(250-400 g body weight). The rats are thyroparathyroidectomized (by thevendor, Taconic Farms) 7 days prior to use. All rats receive areplacement dose of thyroxine every 3 days. On receipt of the rats,circulating ionized calcium levels are measured in whole bloodimmediately after it has been withdrawn by tail venipuncture intoheparinized tubes. Rats are included if the ionized Ca level (measuredwith a Ciba-Corning model 634 calcium pH analyzer) is <1.2 mM/L. Eachrat is fitted with an indwelling venous and arterial catheter for thedelivery of test material and for blood sampling respectively. The ratsare then put on a diet of calcium-free chow and deionized water.Baseline Ca levels are measured and each rat is administered eithercontrol vehicle or human parathyroid hormone 1-34 peptide (hPTH1-34,dose 1.25 ug/kg/h in saline/0.1% bovine serum albumin, Bachem, Ca) or amixture of hPTH1-34 and test material, by continuous intravenousinfusion via the venous catheter using an external syringe pump. Thecalcemic response of each rat is measured at two-hourly intervals duringthe infusion period of 6-8 hours.

Human Osteoclast Resorption and Adhesion Assays

Pit resorption and adhesion assays have been developed and standardizedusing normal human osteoclasts derived from osteoclastoma tissue. Assay1 was developed for the measurement of osteoclast pit volumes by laserconfocal microscopy. Assay 2 was developed as a higher throughput screenin which collagen fragments (released during resorption) are measured bycompetitve ELISA.

Assay 1 (Using Laser Confocal Microscopy)

Aliquots of human osteoclastoma-derived cell suspensions are removedfrom liquid nitrogen strorage, warmed rapidly at 37° C. and washed ×1 inRPMI-1640 medium by centrifugation (1000 rpm, 5 mins at 4° C.).

The medium is aspirated and replaced with murine anti-HLA-DR antibodythen diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30mins on ice and mixed frequently.

The cells are washed ×2 with cold RPMI-1640 followed by centrifugation(1000 rpm, 5 mins at 4° C.) and the cells are then transferred to asterile 15 ml centrifuge tube. The number of mononuclear cells areenumerated in an improved Neubauer counting chamber.

Sufficient magnetic beads (5/mononuclear cell), coated with goatanti-mouse IgG (Dynal, Great Neck, N.Y.) are removed from their stockbottle and placed into 5 ml of fresh medium (this washes away the toxicazide preservative). The medium is removed by immobilizing the beads ona magnet and is replaced with fresh medium.

The beads are mixed with the cells and the suspension is incubated for30 mins on ice. The suspension is mixed frequently.

The bead-coated cells are immobilized on a magnet and the remainingcells (osteoclast-rich fraction) are decanted into a sterile 50 mlcentrifuge tube.

Fresh medium is added to the bead-coated cells to dislodge any trappedosteoclasts. This wash process is repeated×10. The bead-coated cells arediscarded.

The viable osteoclasts are enumerated in a counting chamber, usingfluorescein diacetate to label live cells. A large-bore disposableplastic pasteur pipet is used to add the sample to the chamber.

The osteoclasts are pelleted by centrifugation and the density adjustedto the appropriate number in EMEM medium (the number of osteoclasts isvariable from tumor to tumor), supplemented with 10% fetal calf serumand 1.7 g/liter of sodium bicarbonate.

3 ml aliquots of the cell suspension (per compound treatment) aredecanted into 15 ml centrifuge tubes. The cells are pelleted bycentrifugation.

To each tube, 3 ml of the appropriate compound treatment are added(diluted to 50 uM in the EMEM medium). Also included are appropriatevehicle controls, a positive control (anti-vitronectin receptor murinemonoclonal antibody [87MEM1] diluted to 100 ug/ml) and an isotypecontrol (IgG_(2a) diluted to 100 ug/ml). The samples are incubated at37° C. for 30 mins.

0.5 ml aliquots of the cells are seeded onto sterile dentine slices in a48-well plate and incubated at 37° C. for 2 hours. Each treatment isscreened in quadruplicate.

The slices are washed in six changes of warm PBS (10 ml/well in a 6-wellplate) and then placed into fresh medium containing the compoundtreatment or control samples. The samples are incubated at 37° C. for 48hours.

Tartrate Resistant Acid Phosphatase (TRAP) Procedure (Selective Stainfor Cells of the Osteoclast Lineage)

The bone slices containing the attached osteoclasts are washed inphosphate buffered saline and fixed in 2% gluteraldehyde (in 0.2M sodiumcacodylate) for 5 mins.

They are then washed in water and are incubated for 4 minutes in TRAPbuffer at 37° C. (0.5 mg/ml naphthol AS-BI phosphate dissolved inN,N-dimethylfornamide and mixed with 0.25 M citrate buffer (pH 4.5),containing 10 mM sodium tartrate.

Following a wash in cold water the slices are immersed in cold acetatebuffer (0.1 M, pH 6.2) containing 1 mg/ml fast red garnet and incubatedat 4° C. for 4 minutes.

Excess buffer is aspirated, and the slices are air dried following awash in water.

The TRAP positive osteoclasts (brick red/ purple precipitate) areenumerated by bright-field microscopy and are then removed from thesurface of the dentine by sonication.

Pit volumes are determined using the Nikon/Lasertec ILM21W confocalmicroscope.

Assay 2 (Using an ELISA Readout)

The human osteoclasts are enriched and prepared for compound screeningas described in the initial 9 steps of Assay 1. For clarity, these stepsare repeated hereinbelow.

Aliquots of human osteoclastoma-derived cell suspensions are removedfrom liquid nitrogen strorage, warmed rapidly at 37° C. and washed ×1 inRPMI-1640 medium by centrifugation (1000 rpm, 5 mins at 4° C.).

The medium is aspirated and replaced with murine anti-HLA-DR antibodythen diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30mins on ice and mixed frequently.

The cells are washed ×2 with cold RPMI-1640 followed by centrifugation(1000 rpm, 5 mins at 4° C.) and the cells are then transferred to asterile 15 ml centrifuge tube. The number of mononuclear cells areenumerated in an improved Neubauer counting chamber.

Sufficient magnetic beads (5/mononuclear cell), coated with goatanti-mouse IgG (Dynal, Great Neck, N.Y.) are removed from their stockbottle and placed into 5 ml of fresh medium (this washes away the toxicazide preservative). The medium is removed by immobilizing the beads ona magnet and is replaced with fresh medium.

The beads are mixed with the cells and the suspension is incubated for30 mins on ice. The suspension is mixed frequently.

The bead-coated cells are immobilized on a magnet and the remainingcells (osteoclast-rich fraction) are decanted into a sterile 50 mlcentrifuge tube.

Fresh medium is added to the bead-coated cells to dislodge any trappedosteoclasts. This wash process is repeated ×10. The bead-coated cellsare discarded.

The viable osteoclasts are enumerated in a counting chamber, usingfluorescein diacetate to label live cells. A large-bore disposableplastic pasteur pipet is used to add the sample to the chamber.

The osteoclasts are pelleted by centrifugation and the density adjustedto the appropriate number in EMEM medium (the number of osteoclasts isvariable from tumor to tumor), supplemented with 10% fetal calf serumand 1.7 g/liter of sodium bicarbonate.

In contrast to the method desribed above in Assay 1, the compound arescreened at 4 doses to obtain an IC₅₀, as outlined below:

The osteoclast preparations are preincubated for 30 minutes at 37° C.with test compound (4 doses) or controls.

They are then seeded onto bovine cortical bone slices in wells of a48-well tissue culture plate and are incubated for a further 2 hours at37° C.

The bone slices are washed in six changes of warm phosphate bufferedsaline (PBS), to remove non-adherent cells, and are then returned towells of a 48 well plate containing fresh compound or controls.

The tissue culture plate is then incubated for 48 hours at 37° C.

The supernatants from each well are aspirated into individual tubes andare screened in a competitive ELISA that detects the c-telopeptide oftype I collagen which is released during the resorption process. This isa commercially available ELISA (Osteometer, Denmark) that contains arabbit antibody that specifically reacts with an 8-amino acid sequence(Glu-Lys-Ala-His- Asp-Gly-Gly-Arg) that is present in thecarboxy-terminal telopeptide of the a1-chain of type I collagen. Theresults are expressed as % inhibition of resorption compared to avehicle control.

Human Osteoclast Adhesion Assay

The human osteoclasts are enriched and prepared for compound screeningas described above in the inital 9 steps of Assay 1. For clarity, thesesteps are repeated hereinbelow.

Aliquots of human osteoclastoma-derived cell suspensions are removedfrom liquid nitrogen strorage, warmed rapidly at 37° C. and washed ×1 inRPMI-1640 medium by centrifugation (1000 rpm, 5 mins at 4° C.).

The medium is aspirated and replaced with murine anti-HLA-DR antibodythen diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30mins on ice and mixed frequently.

The cells are washed ×2 with cold RPMI-1640 followed by centrifugation(1000 rpm, 5 mins at 4° C.) and the cells are then transferred to asterile 15 ml centrifuge tube. The number of mononuclear cells areenumerated in an improved Neubauer counting chamber.

Sufficient magnetic beads (5/mononuclear cell), coated with goatanti-mouse IgG (Dynal, Great Neck, N.Y.) are removed from their stockbottle and placed into 5 ml of fresh medium (this washes away the toxicazide preservative). The medium is removed by immobilizing the beads ona magnet and is replaced with fresh medium.

The beads are mixed with the cells and the suspension is incubated for30 mins on ice. The suspension is mixed frequently.

The bead-coated cells are immobilized on a magnet and the remainingcells (osteoclast-rich fraction) are decanted into a sterile 50 mlcentrifuge tube.

Fresh medium is added to the bead-coated cells to dislodge any trappedosteoclasts, This wash process is repeated×10. The bead-coated cells arediscarded.

The viable osteoclasts are enumerated in a counting chamber, usingfluorescein diacetate to label live cells. A large-bore disposableplastic pasteur pipet is used to add the sample to the chamber.

The osteoclasts are pelleted by centrifugation and the density adjustedto the appropriate number in EMEM medium (the number of osteoclasts isvariable from tumor to tumor), supplemented with 10% fetal calf serumand 1.7 g/liter of sodium bicarbonate.

Osteoclastoma-derived osteoclasts are preincubated with compound (4doses) or controls at 37° C. for 30 minutes.

The cells are then seeded onto osteopontin-coated slides (human or ratosteopontin, 2.5 ug/ml) and incubated for 2 hours at 37° C.

Non adherent cells are removed by washing the slides vigorously inphosphate buffered saline and the cells remaining on the slides arefixed in acetone.

The osteoclasts are stained for tattrate-resistant acid phosphatase(TRAP), a selective marker for cells of this phenotype (see steps15-17), and are enumerated by light microscopy. The results areexpressed as % inhibition of adhesion compared to a vehicle control.

Cell Adhesion Assay

Cells and Cell Culture

Human embryonic kidney cells (HEK293 cells) were obtained from ATCC(Catalog No. CRL 1573). Cells were grown in Earl's minimal essentialmedium (EMEM) medium containing Earl's salts, 10% fetal bovine serum, 1%glutamine and 1% Penicillin-Steptomycin.

Constructs and Transfections

A 3.2 kb EcoRI-KpnI fragment of the α_(v) subunit and a 2.4 kb XbaI-XhoIfragment of the β₃ subunit were inserted into the EcoRI-EcoRV cloningsites of the pCDN vector (Aiyar et al., 1994 ) which contains a CMVpromoter and a G418 selectable marker by blunt end ligation. For stableexpression, 80×10⁶ HEK 293 cells were electrotransformed with α_(v)+β₃constructs (20 μg DNA of each subunit) using a Gene Pulser (Hensley etal., 1994 ) and plated in 100 mm plates (5×10⁵ cells/plate). After 48hr, the growth medium was supplemented with 450 μg/mL Geneticin (G418Sulfate, GIBCO-BRL, Bethesda, Md.). The cells were maintained inselection medium until the colonies were large enough to be assayed.

Immunocytochemical Analysis of Transfected Cells

To determine whether the HEK 293 transfectants expressed the vitronectinreceptor, the cells were immobilized on glass microscope slides bycentrifugation, fixed in acetone for 2 min at room temperature and airdried. Specific reactivity with 23C6, a monoclonal antibody specific forthe α_(v)β₃ complex was demonstrated using a standard indirectimmunofluorescence method.

Cell Adhesion Studies

Corning 96-well ELISA plates were precoated overnight at 4° C. with 0.1mL of human vitronectin (0.2 μg/mL in RPMI medium). At the time of theexperiment, the plates were washed once with RPMI medium and blockedwith 3.5% BSA in RPMI medium for 1 hr at room temperature. Transfected293 cells were resuspended in RPMI medium, supplemented with 20 mMHepes, pH 7.4 and 0.1% BSA at a density of 0.5×10⁶ cells/mL. 0.1 mL ofcell suspension was added to each well and incubated for 1 hr at 37° C.,in the presence or absence of various α_(v)β₃ antagonists. Followingincubation, 0.025 mL of a 10% formaldehyde solution, pH 7.4, was addedand the cells were fixed at room temperature for 10 min. The plates werewashed 3 times with 0.2 mL of RPMI medium and the adherent cells werestained with 0.1 mL of 0.5% toluidine blue for 20 min at roomtemperature. Excess stain was removed by extensive washing withdeionized water. The toluidine blue incorporated into cells was elutedby the addition of 0.1 mL of 50% ethanol containing 50 mM HCl. Celladhesion was quantitated at an optical density of 600 nm on a microtiterplate reader (Titertek Multiskan MC, Sterling, Va.).

Solid-Phase α_(v)β₅ Binding Assay

The vitronectin receptor α_(v)β₅ was purified from human placenta.Receptor preparation was diluted with 50 mM Tris-HCl, pH 7.5, 100 mMNaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂ (buffer A) and was immediatelyadded to 96-well ELISA plates at 0.1 ml per well. 0.1-0.2 μg of α_(v)β₃was added per well. The plates were incubated overnight at 4° C. At thetime of the experiment, the wells were washed once with buffer A andwere incubated with 0.1 ml of 3.5% bovine serum albumin in the samebuffer for 1 hr at room temperature. Following incubation the wells wereaspirated completely and washed twice with 0.2 ml buffer A.

In a [³H]-SK&F-107260 competition assay, various concentrations ofunlabeled antagonists (0.001-100 μM) were added to the wells, followedby the addition of 5.0 nM of [³H]-SK&F-107260. The plates were incubatedfor 1 hr at room temperature. Following incubation the wells wereaspirated completely and washed once with 0.2 ml of ice cold buffer A ina well-to-well fashion. The receptors were solubilized with 0.1 ml of 1%SDS and the bound [³H]-SK&F-107260 was determined by liquidscintillation counting with the addition of 3 ml Ready Safe in a BeckmanLS 6800 Liquid Scintillation Counter, with 40% efficiency. Nonspecificbinding of [³H]-SK&F-107260 was determined in the presence of 2 μMSK&F-107260 and was consistently less than 1% of total radioligandinput. The IC₅₀ (concentration of the antagonist to inhibit 50% bindingof [³H]-SK&F-107260) was determined by a nonlinear, least squarescurve-fitting routine, which was modified from the LUNDON-2 program. TheK_(i) (dissociation constant of the antagonist) was calculated accordingto Cheng and Prusoff equation: K_(i)=IC₅₀/(1+L/K_(d)), where L and K_(d)were the concentration and the dissociation constant of[³H]-SK&F-107260, respectively.

Inhibition of RGD-mediated GPIIb-IIIa Binding

Purification of GPIIb-IIIa

Ten units of outdated, washed human platelets (obtained from Red Cross)were lyzed by gentle stirring in 3% octylglucoside, 20 mM Tris-HCl, pH7.4, 140 mM NaCl, 2 mM CaCl₂ at 40° C. for 2 h. The lysate wascentrifuged at 100,000 g for 1 h. The supernatant obtained was appliedto a 5 mL lentil lectin sepharose 4B column (E.Y. Labs) preequilibratedwith 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl₂, 1% octylglucoside(buffer A). After 2 h incubation, the column was washed with 50 mL coldbuffer A. The lectin-retained GPIIb-IIIa was eluted with buffer Acontaining 10% dextrose. All procedures were performed at 4° C. TheGPIIb-IIIa obtained was >95% pure as shown by SDS polyacrylamide gelelectrophoresis.

Incorporation of GPIIb-IIIa in Liposomes

A mixture of phosphatidylserine (70%) and phosphatidylcholine (30%)(Avanti Polar Lipids) were dried to the walls of a glass tube under astream of nitrogen. Purified GPIIb-IIIa was diluted to a finalconcentration of 0.5 mg/mL and mixed with the phospholipids in aprotein:phospholipid ratio of 1:3 (w:w). The mixture was resuspended andsonicated in a bath sonicator for 5 min. The mixture was then dialyzedovernight using 12,000-14,000 molecular weight cutoff dialysis tubingagainst a 1000-fold excess of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mMCaCl₂ (with 2 changes). The GPIIb-IIIa-containing liposomes weecentrifuged at 12,000 g for 15 min and resuspended in the dialysisbuffer at a final protein concentration of approximately 1 mg/mL. Theliposomes were stored at −70° C. until needed.

Competitive Binding to GPIIb-IIIa

The binding to the fibrinogen receptor (GPIIB-IIIa) was assayed by anindirect competitive binding method using [³H]-SK&F-107260 as anRGD-type ligand. The binding assay was performed in a 96-well filtrationplate assembly (Millipore Corporation, Bedford, Mass.) using 0.22 umhydrophilic durapore membranes. The wells were precoated with 0.2 mL of10 μg/mL polylysine (Sigma Chemical Co., St. Louis, Mo.) at roomtemperature for 1 h to block nonspecific binding. Various concentrationsof unlabeled benzazepines were added to the wells in quadruplicate.[³H]-SK&F-107260 was applied to each well at a final concentration of4.5 nM, followed by the addition of 1 μg of the purified plateletGPIIb-IIIa-containing liposomes. The mixtures were incubated for 1 h atroom temperature. The GPIIb-IIIa-bound [³H]-SK&F-107260 was seperatedfrom the unbound by filtration using a Millipore filtration manifold,followed by washing with ice-cold buffer (2 times, each 0.2 mL). Boundradioactivity remaining on the filters was counted in 1.5 mL Ready Solve(Beckman Instruments, Fullerton, Calif.) in a Beckman LiquidScintillation Counter (Model LS6800), with 40% efficiency. Nonspecificbinding was determined in the presence of 2 μM unlabeled SK&F-107260 andwas consistently less than 0.14% of the total radioactivity added to thesamples. All data points are the mean of quadruplicate determinations.

Competition binding data were analyzed by a nonlinear least-squarescurve fitting procedure. This method provides the IC50 of theantagonists (concentration of the antagonist which inhibits specificbinding of [³H]-SK&F-107260 by 50% at equilibrium). The IC50 is relatedto the equilibrium dissociation constant (Ki) of the antagonist based onthe Cheng and Prusoff equation: Ki=IC50/(1+L/Kd), where L is theconcentration of [³H]-SK&F-107260 used in the competitive binding assay(4.5 nM), and Kd is the dissociation constant of [³H]-SK&F-107260 whichis 4.5 nM as determined by Scatchard analysis.

The efficacy of the compound of formula (I) alone or in combination withan antineoplastic agent may be determined using several transplantablemouse tumor models. See U.S. Pat. Nos. 5,004,758 and 5,633,016 fordetails of these models

The examples which follow are intended in no way to limit the scope ofthis invention, but are provided to illustrate how to make and use thecompound of this invention. Many other embodiments will be readilyapparent to those skilled in the art.

EXAMPLES General

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded at 300MHz, and chemical shifts are reported in parts per million (δ) downfieldfrom the internal standard tetramethylsilane (TMS). Abbreviations forNMR data are as follows: s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet, dd=doublet of doublets, dt=doublet of triplets,app=apparent, br=broad. J indicates the NMR coupling constant measuredin Hertz. CDCl₃ is deuteriochloroform, DMSO-d₆ ishexadeuteriodimethylsulfoxide, and CD₃OD is tetradeuteriomethanol. Massspectra were obtained using electrospray (ES) ionization techniques.Elemental analyses were performed by Quantitative Technologies Inc.,Whitehouse, N.J. Melting points were obtained on a Thomas-Hoover meltingpoint apparatus and are uncorrected. All temperatures are reported indegrees Celsius. Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254thin layer plates were used for thin layer chromatography. Flashchromatography was carried out on E. Merck Kieselgel 60 (230-400 mesh)silica gel. Analytical and preparative HPLC were carried out on Beckmanchromatographs. ODS refers to an octadecylsilyl derivatized silica gelchromatographic support. YMC ODS-AQ® is an ODS chromatographic supportand is a registered trademark of YMC Co. Ltd., Kyoto, Japan. PRP-1® is apolymeric (styrene-divinylbenzene) chromatographic support, and is aregistered trademark of Hamilton Co., Reno, Nev. Celite® is a filter aidcomposed of acid-washed diatomaceous silica, and is a registeredtrademark of Manville Corp., Denver, Colo.

Preparation 1 Preparation of2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethanol

a) 2-Methyl-8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthyridine

A mixture of 2-methyl-1,8-naphthyridine (J. Chem. Soc. (C) 1966, 315;5.13 g, 35.58 mmole), 10% Pd/C (1.14 g, 1.07 mmole), and absolute EtOH(70 mL) was deoxygenated through three evacuation/H₂ purge cycles, thenwas stirred briskly under a balloon of H₂. After 18.5 hr, the mixturewas filtered through celite®, and the filter pad was washed sequentiallywith absolute EtOH and EtOAc. The filtrate was concentrated to dryness,and the residue was reconcentrated from EtOAc to leave an off-whitesolid (5.25 g).

A solution of the above material (5.25 g), di-tert-butyl dicarbonate(15.53 g, 71.16 mmole), and CH₂Cl₂ (10 mL) was concentrated on therotavap to remove the solvent, and the oily residue was heated under N₂in an oil bath set at 55-60° C. After 45 hr, the reaction was cooled toRT, and the residue was flash chromatographed on silica gel (40%EtOAc/hexanes). The title compound (4.90 g, 55%) was obtained as a lightyellow solid: ¹H NMR (300 MHz, CDCl₃) δ7.27 (d, J=7.6 Hz, 1H), 6.81 (d,7.6 Hz, 1H), 3.69 -3.79 (m, 2H), 2.65-2.75 (m, 2H), 2.48 (s, 3H),1.83-1.98 (m, 2H), 1.52 (s, 9H), MS (ES) m/e 249 (M+H)⁺.

b) Ethyl[8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl]acetate

To a solution of diisopropylamine (7.24 mL, 55.3 mmole) in dry TBF (50mL) was added n-BuLi (2.5 M in hexanes, 22 mL, 55.3 mmole) dropwise at0° C. After 15 min, this solution was added dropwise to a solution of2-methyl-8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthyridine(4.9 g, 19.7 mmole) and diethylcarbonate (8.86 mL, 73.0 mmole) in dryTHF (50 mL) at −78° C. After 30 min, the mixture was quenched withsaturated NH₄Cl (100 mL), warmed to RT, and extracted with EtOAc (3×200mL). The combined organic extracts were dried over MgSO₄, filtered, andconcentrated under reduced pressure. The residue was chromatographed onsilica gel (40% EtOAc/hexanes) to give the title compound (5.72 g, 91%)as a light yellow oil: MS (ES) m/e 321 (M+H)⁺.

c) 2-(5,6,7,8-Tetrahydro-1,8-naphthyridin-2-yl)-1-ethanol

To a solution of ethyl[8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl]acetate(5.72 g, 17.85 mmole) in dry THF (80 mL) at RT was added LiBH₄ (2.0 M inTHF, 10.7 mL, 21.42 mmole), and the resulting mixture was heated toreflux. After 18 hr, the mixture was cooled to 0° C. and carefullyquenched with H₂O (100 mL). After 10 min, the mixture was extracted withEtOAc (3×100 mL). The combined organic extracts were dried over MgSO₄,filtered, and concentrated under reduced pressure.

The above residue (4.9 g) was dissolved in CH₂Cl₂ (10 mL). To this wasadded 4 N HCl in dioxane (20 mL) all at once at RT. After 4, the mixturewas concentrated under reduced pressure. The residue was taken up in a1:1 mixture of 1.0 N NaOH and saturated NaCl (100 mL) and extracted withCH₂Cl₂ (3×100 mL). The combined organic extracts were dried over MgSO₄,filtered, and concentrated under reduced pressure. The residue waschromatographed on silica gel (10% MeOH in 1:1 EtOAc/CHCl₃) to give thetitle compound (2.09 g, 66%) as a yellow solid: MS (ES) m/e 179 (M+H)⁺.

Preparation 2 Preparation of ethyl(±)-10,11-dihydro-3-hydroxy-5H-dibenzo[a,d]cycloheptene-10-acetate

a) 6-Methoxy-1-phenylindene

A solution of 3.0 M phenylmagnesium bromide in Et₂O (680 mL, 2.04 mole)under argon at ambient temperature was diluted with Et₂O (700 mL) withstirring, and a solution of 6-methoxy-1-indanone (277 g, 1.71 mole) inTHF (1400 mL) was added dropwise over 1 hr. The reaction mixture wasstirred for 2 h at ambient temperature and then was poured with stirringinto saturated NH₄Cl (2.8 L). H₂O (1.4 L) was added, and the organicphase separated. The aqueous phase was extracted with Et₂O (2×1 L), andthe combined organic extracts were concentrated to give crude6-methoxy-1-phenyl-1-indanol (445 g) as a brown oil. This oil wasdissolved in toluene (2.5 L), and p-toluenesulfonic acid monohydrate(12.3 g, 0.065 mole) was added. The solution was stirred and heated atreflux for 16 hr using a Dean-Stark trap with a condenser. H₂Ocollection was minimal after 2 h and totaled 28 mL. The solution wascooled and extracted sequentially with 5% aqueous Na₂CO₃ (1 L) and H₂O(2×1 L). The organic layer was concentrated to give a dark brown oil(400 g). This oil was distilled under vacuum to give the title compound(298.2 g, 79%) as a yellow oil: bp 152-190° C./2.0 Torr; TLC (10%EtOAc/hexanes) R_(f)0.75.

b) 2-Benzoyl-4-methoxyphenylacetic Acid

Acetone (4.2 L) was chilled to 10° C., and a solution of6-methoxy-1-phenylindene (271 g, 1.22 mole) in acetone (1.8 L) was addedover 1.5 hr concurrently with Jones reagent (1.8 L, prepared from CrO₃(470 g, 4.70 mole), H₂O (1 L), and conc H₂SO₄ (405 mL)). 4% Aqueous OsO₄(153 mL) was added to the resulting mixture in two portions, one at theonset of addition and the second at the mid-point of the addition,maintaining the temperature of the reaction mixture below 15° C.Following the addition, the reaction mixture was warmed to 22° C. andstirred for 1.5 h, during which time a mild exotherm increased thetemperature to 28° C. The reaction mixture was then cooled to below 20°C. and isopropanol (1 L) was added, dropwise initially and rapidly afterthe initial exotherm diminished. Stirring became difficult during thisphase. The temperature reached 32° C. during the isopropanol addition.H₂O (2 L) was added and the mixture was transferred to a separatoryfunnel. Additional H₂O was added to dissolve the precipitated chromousacid, and the mixture was extracted with CH₂Cl₂ (2 L). The organic(upper) layer was separated and the aqueous phase was extracted withCH₂Cl₂ (2×1 L). The combined CH₂Cl₂ extracts were washed sequentiallywith H₂O (2 L) and saturated brine (2 L), and then were concentrated togive a moist gray solid (416 g). This was triturated with a mixture ofacetone and EtOAc and filtered and dried to give the title compound(225.4 g, 71%) as an off-white solid: mp 158-159° C.

c) 2-Benzyl-4-methoxyphenylacetic Acid

2-Benzoyl-4-methoxyphenylacetic acid (215.5 g, 0.80 mole) was dividedinto two equal portions, and each was dissolved in glacial AcOH (1.5 L)in a 2.5 L pressure bottle. 5% Pd/C (10 g, 0.0048 mole) was added toeach, and each mixture was shaken at ambient temperature under hydrogenon a Parr apparatus. After 2.5 hr, the mixtures were filtered to removethe catalyst, and the filter pads were washed with EtOAc. The combinedfiltrates were concentrated to give the title compound (215 g,quantitative) as a heavy yellow oil which crystallized on standing: ¹HNMR (250 MHz, CDCl₃) δ7.05-7.35 (m, 6H), 6.77 (dd, J=8.3, 2.7 Hz, 1H),6.71 (d, J=2.7 Hz, 1H), 4.00 (s, 2H), 3.76 (s, 3H), 3.54 (s, 2H).

d) 10,11-Dihydro-3-methoxy-5H-dibenzo[a,d]cyclohepten-10one

A solution of 2-benzyl-4methoxyphenylacetic acid (215 g of crudematerial that contained 204.6 g (0.80 mole) of pure material) in CH₂Cl₂(1 L) was stirred under argon at ambient temperature, and DMF (1 mL) wasadded, followed by oxalyl chloride (400 mL, 4.59 mole). The oxalylchloride was added over 1 hr, dropwise initially to control the vigorousgas evolution. The solution was stirred for 16 h at ambient temperatureand then was concentrated to give the crude acid chloride (207.7 g,0.756 mol, 95%) as a yellow liquid. This liquid was dissolved in CH₂Cl₂to a total volume of 500 mL, and the solution and AlCl₃ (100.8 g, 0.756mol) were added concurrently over 1 hr to CH₂Cl₂ (3.7 L) with stirringunder argon at ambient temperature. The temperature was 28° C. at thecompletion of the addition. The reaction mixture was stirred for 16 h atambient temperature, during which time a solid precipitated. H₂O (1 L)was added, initially dropwise, over a period of 30 min. The mixture wasthen separated and the organic phase was washed sequentially with H₂O (1L) and 5% aqueous NaHCO₃ (1 L). The CH₂Cl₂ solution was thenconcentrated to give a yellow solid (175.3 g). Recrystallization fromEtOAc/hexane gave the title compound (128 g, 71%): mp 107-109° C.

e) Ethyl (±)-10,11-dihydro-10-hydroxy-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate

A 1.0 M solution of lithium bis(trimethylsilyl)amide in hexanes (1282mL, 1.282 mole) was added to THF (4.0 L) at −70° C. under argon, thenEtOAc (146 mL, 1.49 mole) was added dropwise over 20 min. The reactionmixture was allowed to stir for 15 min, thenN,N,N′,N′-etramethylethlylenediamine (378 mL, 2.5 mole) was added over20 min. The reaction mixture was stirred for 10 min, then a solution of10,11-dihydro-3-methoxy-5H-dibenzo[a,d]cyclohepten-10-one (119.2 g, 0.50mol) in anhydrous THF (1.26 L) was added dropwise over 40 min. Thetemperature was maintained below −65° C. during all of these additions.The reaction mixture was stirred for 20 min at −65 to −70° C. and thenwas poured into saturated aqueous NH₄Cl (6.2 L) with vigorous stirring.The organic layer was separated and the aqueous phase was extracted withEtOAc (2×1 L). The combined organic extracts were washed with H₂O (2×1L) and then were concentrated to give a light brown oil (175 g).Thin-layer chromatography (20% EtOAc/hexanes) showed R_(f)0.5 major(desired product) and R_(f)0.7 minor (recovered ketone). The crudeproduct was chromatographed on silica gel (2 kg, 10% EtOAc/hexanes) toafford the title compound (101 g, 61%) as a yellow oil: ¹H NMR (250 MHz,CDCl₃) δ7.63 (d, J=7.7 Hz, 1H), 7.00-7.30 (m, 4H), 6.80 (d, J=2.6 Hz,1H), 6.69 (dd, J=8.2, 2.6 Hz, 1H), 3.95-4.35 (m, 2H), 4.07 (s, 2H), 3.76(s, 3H), 3.68 (s, 1H), 3.64 (d, J=14.2 Hz, 1H), 3.35 (d, J=14.2 Hz, 1H),2.79 (d, J=16.0 Hz, 1H), 2.66 (d, J=16.0 Hz, 1H), 1.22 (t, J=7.2 Hz,3H).

f) Ethyl (±)-10,11-dihydro-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate

Ethyl(±)-10,11-dihydro-10hydroxy-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate(101 g, 0.31 mole) was dissolved in glacial acetic acid (1.8 L) and 12 NHCl (28.5 mL, 0.34 mole) was added. The mixture was placed in a 2.5 Lpressure both containing 5% Pd/C (20 g, 0.0094 mole), and the resultingmixture was shaken at 35° C. under hydrogen on a Parr hydrogenationapparatus equipped with a jacket heater. After 18 hr, the reaction wascooled to ambient temperature, and the catalyst was removed byfiltration. The filtrate was concentrated to give a light yellow oil(85.1 g). This was chromatographed on silica gel (2 kg, step-gradientwith 5% to 10% EtOAc/hexanes) to afford the title compound (69.1 g, 72%)as an oil: ¹H NMR (250 MHz, CDCl₃) δ7.05-7.22 (m, 4H), 7.01 (d, J=8.2Hz, 1H), 6.76 (d, J=2.7 Hz, 1H), 6.67 (dd, J=8.2, 2.7 Hz, 1H), 4.30 (d,J=15.0 Hz, 1H), 4.11-4.25 (m, 2H), 3.85 (d, J=15.0 Hz, 1H), 3.70-3.90(m, 1H), 3.77 (s, 3H), 3.31 (dd, J=15.0, 4.1 Hz, 1H), 2.93 (dd, J=15.0,9.2 Hz, 1H), 2.64 (dd, J=15.6, 5.0 Hz, 1H), 2.52 (dd, J=15.6, 9.3 Hz,1H), 1.27 (t, J=7.1 Hz, 3H).

g) Ethyl(±)-10,11-dihydro-3-hydroxy-5H-dibenzo[a,d]cycloheptene-10-acetate

A solution of ethyl(±)-10,11-dihydro-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate (8.5g, 0.027 mole) in CH₂Cl₂ (150 mL) was chilled to −10° C. with stirringunder argon. Ethanethiol (10.7 mL, 0.144 mole) was added, followed byAlCl₃ (20.6 g, 0.154 mole) in two portions over 15 min. An exothermincreased the temperature to 0° C. following the additions, and thetemperature was then increased to 25° C. using a water bath. Thereaction mixture was stirred at 25 to 30° C. for 2.25 hr, at which pointit was poured into ice-H₂O. The organic layer was separated, methanol(100 mL) was added, and the mixture was extracted with CH₂Cl₂ (2×50 mL).The combined CH₂Cl₂ extracts were washed with H₂O (250 mL) and then wereconcentrated to give a viscous oil (8.6 g). This was taken up in Et₂O(150 mL) and the ether was boiled off while replacing it with hexane.The desired phenol first separated as an oil which crystallized onstirring at ambient temperature. Two crops of solid were collected toafford the title compound (7.1 g, 89%): mp 110-112° C.

Preparation 3 HPLC Separation of the Enantiomers of ethyl(±)-10,11-dihydro-3-hydroxy-5H-dibenzo[a,d]cycloheptene-10-acetate

a) Ethyl(R)-(+)-10,11-dihydro-3-hydroxy-5H-dibenzo[a,d]cycloheptene-10-acetateand ethyl (S)-(−)-10,11-dihydro-3-hydroxy-5-dibenzo[a,d]cycloheptene-10-acetate

Ethyl (±)-10,11-dihydro-3-hydroxy-5H-dibenzo[a,d]cycloheptene-10-acetatewas resolved into its enantiomers using the following conditions: DaicelChiralcel OJ® column (21.2×250 mm), 20% ethanol in hexane mobile phase,15 mL/min flow rate, uv detection at 254 nm, 140 mg injection; t_(R) forethyl(S)-(−)-10,11-dihydro-3-hydroxy-5-dibenzo[a,d]cyclohexene-10-acetate=10.4min.; t_(R) for ethyl(R)-(+)-10,11-dihydro-3-hydroxy-5H-dibenzo[a,d]cyclohexene-10-acetate=13.1min.

Preparation 4 Preparation of10,11-Dihydro-3-methoxy-5H-dibenzo[a,d]cyclohepten-10-one

a) 2-Benzyl-4-methoxyphenylacetic Acid

A solution of 2-benzoyl-4-methoxyphenylacetic acid (13.0 g, 0.048 mol),prepared by the method of J. Med. Chem. 1981, 24, 998, in glacial aceticacid (600 mL) was treated under argon with 4.3 g. of 10% Pd/C andhydrogenated at 50 psi for 17 hours. The mixture was filtered usingcelite® and the filtrate was concentrated and reconcentrated fromtoluene and methylene chloride to give 14.2 of the title compound: ¹HNMR (400 MHz, CDCl₃) δ3.52 (s, 2H), 3.75 (s, 3H), 4.0 (s, 3H), 6.7 (m,2H), 7.15 (m, 6H).

b) 10,11-Dihydro-3-methoxy-5H-dibenzo[a,d]cyclohepten-10-one

A solution of 2-benzyl-4-methoxyphenylacetic acid (14.2 g, 0.055 m) inbenzene (120 mL) and thionyl chloride (28 mL) was refluxed for 1 hourand concentrated. The acid chloride was dissolved in dry methylenechloride (40 mL), and the solution was added dropwise under argon to asolution of AlCl₃ (14.7 g, 0.11 mol) in methylene chloride (600 mL). Thereaction was stirred under an argon atmosphere for 2.5 hours at roomtemperature, then was quenched with ice-water (200 mL). The layers wereseparated, and the organic phase was washed sequentially with 10% NaOHsolution, water, and dil. HCl. The resulting solution was diluted withether (200 mL), dried over MgSO₄, and concentrated. The solid residuewas triturated with ether/hexane (1:1) and 9.35 g of the title compoundwas collected by filtration: Mp 105-106° C.; ¹H NMR (400 MHz, CDCl₃)δ3.72 (s, 3H), 4.1 (s, 2H), 4.2 (s, 2H), 6.7 (d, 1H), 6.82 (s, 1H), 7.30(m, 4H), 8.1 (d, 1H).

Preparation 5 Preparation of ethyl(±)-10,11-dihydro-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate

a) Ethyl (±) 3-(3-methoxyphenyl)indeneacetate

To a cold solution of 3-(3-methoxyphenyl)indene (4 g, 18 mmol), preparedby the method of J. Med. Chem. 1981, 24, 998, in THF (15 mL) at 0° C.was added dropwise a solution of LiN(TMS)₂ (20 mL, 1M in THF) over 5min. The resulting solution was added dropwise to a solution of ethylbromoacetate (3.34 g, 20 mmol) in THF (15 mL) at −78° C. over 30 min.After 2.5 h, the mixture was quenched with saturated ammonium chloridesolution and the layers were separated. The organic layer was dried overMgSO₄ and concentrated to give the crude product which was purified bycolumn chromatography (SiO₂/2-4% EtOAc/hexane) to give title compound(1.1 g): ¹H NMR (400 MHz, CDCl₃) δ1.30 (t, 3H), 2.50 (m, 1H), 2.85 (m,1H), 3.85 (s, 3H), 4.0 (m, 1H), 4.20 (q, 2H), 6.6 (s, 1H), 6.9 (m, 1),7.2 (s, 1H), 7.35 (m, 6H).

b) Ethyl (±) 3-[(3-methoxybenzoyl)]phenylsuccinate

A solution of ethyl (±) 3-(3-methoxyphenyl)indeneacetate (1.1 g, 3.6mmol) in acetone (30 mL) was treated with 4% aqueous solution of osmiumtetroxide (0.5 mL) followed by a dropwise addition of 1.2 M Jonesreagent (5 mL, 6 mmol) according to the literature procedure (J. Org.Chem. 1993, 58, 4745). After stirring overnight at room temperature, thedark reaction mixture was quenched with isopropanol (2.5 mL), followedby sodium bisulfite (0.9 g) and water (30 mL). The product was extractedwith ethyl acetate, washed with brine, dried over MgSO₄, andconcentrated to give a solid residue. Trituration with 1:1 ether/hexanegave 0.76 g of the title compound: ¹H NMR (400 MHz, CDCl₃) δ1.18 (t,3H), 2.90 (m, 1H), 3.3 (m, 1H), 3.92 (s, 3H), 4.1 (q, 2H), 4.4 (m, 1H),4.4 (d, 1H), 7.25 (m, 2H), 7.5 (m, 6H).

c) Ethyl (±) 3-[(3-methoxybenzyl)]phenylsuccinate

A mixture of ethyl (±) 3-[(3-methoxybenzoyl)]phenylsuccinate (0.76 g.,2.1 mmol) and 10% Pd/C (0.6 g) in glacial acetic acid (35 mL) washydrogenated at 50 psi for 17 hours. The mixture was filtered usingcelite® and the filter pad was washed with acetic acid. The filtrate wasconcentrated and reconcentrated from toluene and methylene chloride togive 0.65 g of the title compound: ¹H NMR (400 MHz, CDCl₃) δ1.20 (t,3H), 2.20 (m, 1H), 3.0 (m, 1H), 3.74 (s, 3H), 4.1 (q, 2H), 4.18 (q, 2H),4.4 (d, 1H), 6.2 (m, 2H), 7.22 (m, 6H).

d) Ethyl(±)-10,11-dihydro-3-methoxy-11-oxo-5-dibenzo[a,d]cycloheptene-10-acetate

To a magnetically stirred solution of ethyl (±)3-[(3-methoxybenzyl)]phenylsuccinate (0.65 g, 1.9 mmol) in dry methylenechloride (10 mL) were added DMF (0.2 mL) and oxalyl chloride (0.2 mL,2.28 mmol). After 1.5 h, the solution was added dropwise to a suspensionof aluminum chloride (0.6 g, 4.5 mmol) in dry methylene chloride (15mL). The mixture was quenched after 2 h with ice water, the layers wereseparated, and the aqueous layer was extracted with methylene chloride.The combined organic layers were dried over MgSO₄ and concentrated. Theresidue was purified by column chromatography (SiO₂/2-4% EtOAc/hexane)to give title compound (0.3 g): ¹H NMR (400 MHz, CDCl₃) δ1.28 (t, 3H),2.88 (m, 1H), 3.55 (m, 1H), 3.84 (s, 3H), 3.88 (d, 1H), 4.18 (q, 2H),4.85 (d, 1H), 4.95 (m, 1H), 5.8 (m, 2H), 7. 22(m, 4H), 8.1 (s, 1H).

e) Ethyl(±)-10,11-dihydro-3-methoxy-5H-dibenzo[a,d]cycloheptene-10-acetate

A mixture of ethyl(±)-10,11-dihydro-3-methoxy-11-oxo-5H-dibenzo[a,d]cycloheptene-10-acetate(0.3 g., 0.93 mmol) and 10% Pd/C (0.3 g) in glacial acetic acid (25 mL)was hydrogenated at 50 psi for 18 hours. The mixture was filtered usingcelite® and washed with acetic acid. The filtrate was concentrated andreconcentrated from toluene and methylene chloride to give 0.25 g of thetitle compound: ¹H NMR (400 MHz, CDCl₃) δ1.28 (t, 3H), 2.60 (m, 2H),2.90 (m, 1H), 3.30 (m, 1H), 3.80 (s, 3H), 3.85 (d, 1H), 4.18 (q, 2H),4.30 (d, 1H), 6.70 (m, 2H), 7.0 (d, 1H), 7.22 (m, 4H).

The following example illustrates a method for preparing thebiologically active compound of this invention from intermediatecompounds such as described in the foregoing Preparations.

Example 1 Preparation of(S)-10,11-dihydro-3-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethoxy]-5H-dibenzo[a,d]cycloheptene-10-aceticAcid

a) Ethyl(S)-10,11-dihydro-3-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethoxy]-5H-dibenzo[a,d]cycloheptene-10-acetate

To a solution of ethyl (S)-10,11-dihydro-3-hydroxy-5H-dibenzo[a,d]cycloheptene-10-acetate (200 mg, 0.67mmole), 2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1 -ethanol (241 mg,1.35 mmole), and PPh₃ (354 mg, 1.35 mmole) in dry THF (5 mL) was addeddiisopropyl azodicarboxylate (0.27 mL, 1.35 mmole) at 0° C. The mixturewas allowed to warm to RT as the bath warmed. After 18 hr, the mixturewas concentrated under reduced pressure. The residue was chromatographedon silica gel (1:4.5 hexanes/Et₂O) to give the title compound (94 mg,31%) as a clear oil: MS (ES) m/e 457 (M+H)⁺.

b)(S)-10,11-Dihydro-3-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethoxy]-5H-dibenzo[a,d]cycloheptene-10-aceticacid

To a solution of ethyl(S)-10,11-dihydro-3-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethoxy]-5H-dibenzo[a,d]cycloheptene-10-acetate(131 mg, 0.29 mmole) in THF/H₂O (2 mL) was added 1.0 N LiOH (0.43 mL,0.43 mmole), and the mixture was heated to 50° C. After 18 hr, themixture was cooled to RT and washed with Et₂O (2×2 mL). The aqueouslayer was acidified to pH 6 using 10% HCl. The resulting milky solutionwas passed through a C-18 bond-elute column (gradient elution: H₂O, then20% CH₃CN/H₂O, then CHCl₃ as eluent). Fractions containing the productwere concentrated under reduced pressure to give the title compound (30mg, 24%) as a white powder: MS (ES) m/e 429 (M+H)⁺. Anal. Calcd forC₂₇H₂₈N₂O₃.0.95 HCl: C, 70.02; H, 6.30; N, 6.05. Found: C, 70.01; H,6.33; N, 5.71.

Example 2

Parenteral Dosage Unit Composition

A preparation which contains 20 mg of the compound of Example 1 as asterile dry powder is prepared as follows: 20 mg of the compound isdissolved in 15 mL of distilled water. The solution is filtered understerile conditions into a 25 mL multi-dose ampoule and lyophilized. Thepowder is reconstituted by addition of 20 mL of 5% dextrose in water(D5W) for intravenous or intramuscular injection. The dosage is therebydetermined by the injection volume. Subsequent dilution may be made byaddition of a metered volume of this dosage unit to another volume ofD5W for injection, or a metered dose may be added to another mechanismfor dispensing the drug, as in a bottle or bag for IV drip infusion orother injection-infusion system.

Example 3

Oral Dosage Unit Composition

A capsule for oral administration is prepared by mixing and milling 50mg of the compound of Example 1 with 75 mg of lactose and 5 mg ofmagnesium stearate. The resulting powder is screened and filled into ahard gelatin capsule.

Example 4

Oral Dosage Unit Composition

A tablet for oral administration is prepared by mixing and granulating20 mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of thecompound of Example 1 with a 10% gelatin solution. The wet granules arescreened, dried, mixed with 10 mg starch, 5 mg talc and 3 mg stearicacid; and compressed into a tablet.

The above description fully discloses how to make and use the presentinvention. However, the present invention is not limited to theparticular embodiments described hereinabove, but includes allmodifications thereof within the scope of the following claims. Thevarious references to journals, patents and other publications which arecited herein comprises the state of the art and are incorporated hereinby reference as though fully set forth.

2 1 7 DNA Homo sapien 1 arggyas 7 2 17 DNA Homo Sapien 2 guysaahsasgygyarg 17

What is claimed is:
 1. A compound according to formula (I):

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition which comprises a compound according to claim 1 and apharmaceutically acceptable carrier.
 3. A pharmaceutical compositionwhich comprises a compound according to claim 1, an antineoplastic agentand a pharmaceutically acceptable carrier.
 4. The pharmaceuticalcomposition according to claim 3 wherein the antineoplastic agent istopotecan.
 5. The pharmaceutical composition according to claim 3wherein the antineoplastic agent is cisplatin.
 6. A method of treating adisease state in which antagonism of the α_(v)β₃ receptor is indicatedwhich comprises administering to a subject in need thereof a compoundaccording to claim
 1. 7. A method of treating a disease state in whichantagonism of the α_(v)β₅ receptor is indicated which comprisesadministering to a subject in need thereof a compound according toclaim
 1. 8. A method of treating osteoporosis which comprisesadministering to a subject in need thereof a compound according toclaim
 1. 9. A method for inhibiting angiogenesis which comprisesadministering to a subject in need thereof a compound according toclaim
 1. 10. A method for inhibiting tumor growth or tumor metastasiswhich comprises administering to a subject in need thereof a compoundaccording to claim
 1. 11. A method of treating atherosclerosis orrestenosis which comprises administering to a subject in need thereof acompound according to claim
 1. 12. A method of treating inflammationwhich comprises administering to a subject in need thereof a compoundaccording to claim
 1. 13. A method of inhibiting tumor growth whichcomprises administering stepwise or in physical combination a compoundaccording to claim 1 and an antineoplastic agent.
 14. The methodaccording to claim 13 wherein the antineoplastic agent is topotecan. 15.The method according to claim 13 wherein the antineoplastic agent iscisplatin.
 16. A compound according to formula (II):

or a pharmaceutically acceptable salt thereof.
 17. A process forpreparing a compound of the formula (I) as defined in claim 1, whichprocess comprises reacting a compound of formula (III):

with 2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-ethanol in areaction mediated by the complex formed between diisopropylazodicarboxylate and triphenylphosphine, followed by ester hydolysisusing aqueous base.